Delivery method

ABSTRACT

A method of altering cells comprising culturing them in a growth factor gradient, wherein said gradient is provided by a polyhedra delivery system (PODS) that releases the growth factor to set up the gradient.

FIELD OF THE INVENTION

This invention relates to a method of culturing cells.

BACKGROUND

The development and maintenance of the organisms requires many complexinteractions between cells and extracellular matrix (ECM) components.Control of the cellular microenvironment is also important for assuringfunctionality of tissue engineered organ substitutes. The use of ECMmimics, which utilize collagen gel compaction, electromagnetic fields,electrospinning of nanofibers, mechanical stimulation andmicrostructured culture plates for artificial guidance of cells, haveall been explored.

SUMMARY

The inventors have found that a polyhedron-based delivery system whichreleases growth factor (Polyhedra Delivery System, PODS) provides astable physiologically relevant gradient of growth factor. Theinventors' work investigates the activity of a growth factor gradientgenerated by a PODS which is set up by sustained release over a periodof time, and is able to direct changes in relevant growth factorsensitive cells. The inventors have surprisingly found that using a PODSin this way allows differentiated cells with desired properties, such asdirectional growth, to be produced in the absence of extracellularmatrix (ECM). Part of their work concerns preparation of an unbranchedchain of neurons connected by axons.

Accordingly a first aspect of the invention provides a method ofaltering cells comprising culturing them in a growth factor gradient,wherein said gradient is provided by a polyhedra delivery system (PODS)that releases the growth factor to set up the gradient.

A second aspect of the present invention provides PODS as a therapeuticagent for use in a method of therapy.

A third aspect of the invention provides cells altered by the methods ofthe invention, optionally with a delivery vehicle, for use in a methodof therapy.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Construction of NGF Polyhedra. Full length and mature NGF (fullNGF and mature NGF, respectively) were fused with either a H1 or VP3tag, and each recombinant NGF fusion protein was encapsulated intopolyhedra.

FIG. 2. PODS EGFP and PODS NGF were mixed and spotted. PC12 cells wereseeded and incubated for 5 days (left). Green fluorescence was scannedwithin the white box (middle) and measured by counting the number ofpixels that exhibited green fluorescence (right). The aligned PC12 cellswere detected by the fluorescence from PODS field. The dotted lineindicates the extent of the EGFP gradient

FIG. 3. PC12 cells were incubated with PODS H1/full NGF. PC12 cells wereincubated with PODS H1/full NGF. Expression of tau and neurofilamentwhich was induced by PODS NGF was analyzed by immunofluorescencecytochemistry. Nuclei were stained with propidium iodide (PI).Connection of PC12 cells via the extended axon was observed. Theextended axon which was induced by PODS NGF was detected by expressionof neurofilament. The solid box shows the connection between theextended axon and the growth cone-like structure.

DETAILED DESCRIPTION OF THE INVENTION Terminology

The terms ‘PODS’, ‘microcrystal’ and ‘crystal’ are used interchangeablyherein. However it should be understood that in embodiments which referto ‘microcrystal’ or ‘crystal’ non-crystalline forms of PODS can also beused.

Characteristics of the Invention

When used in culture systems growth factors can be rapidly andhomogenously diffused, but the imbalance and asymmetry of growth factorsis important for the development of certain cells. The invention showsthese complex phenomena can be reproduced in vitro by a PODS thatreleases a growth factor. The invention concerns altering cells by meansof a gradient of growth factor generated by a PODS. The altering of thecells which occurs in the method of the invention may be due to one partof the cell being in contact with the growth factor at a differentconcentration from another part of the cell. It may be due to one cellbeing in communication or contact with another cell in contact with thegrowth factor at a different concentration. Through this mechanism thegradient is able to impart certain characteristic to the cell whichwould not be possible where the growth factor was present at ahomogenous concentration, for example at the same concentration at eachpoint of the cell surface. Further the fact that the gradient isunchanging is also an important in imparting the desired characteristicto the cell.

Culturing

The invention concerns culturing cells, which typically comprisesplacing them in conditions where they grow and/or differentiate. Theconditions may be in vivo or in vitro, such as in a human or animalbody.

Whilst the features of the invention will be described with reference toits in vitro embodiments these features may also apply to the in vivoembodiments as appropriate.

Culturing in vitro is typically in an aqueous medium. Optionally theculturing takes place in a medium comprising aqueous gel. The culturingmay take place in a vessel, optionally a dish. The culturing may or maynot take place on a coverslip, optionally in a culture medium in contactwith a coverslip.

The culturing may be in any suitable system, for example a 2-dimensionalor 3-dimensional system. The culturing may in static conditions, forexample in which there is no flow of medium and/or there is no change inmedium. In preferred embodiments medium is not changed for at least 10hours, for example for at least 20, 30, 50, 100 or 500 hours.

The culturing will preferably take place in a medium in which is able tosustain the cells and/or allow them to grow. The medium typicallycomprises one or more nutrients. The medium may or may not beserum-free. The medium may comprise DMEM or neurobasal.

Cells

As used herein the term cells typically refers to eukaryotic cells,preferably human or animal cells, such as mammalian or avian cells.Optionally the cells described herein are neuronal cells. Preferably thecells are PC12 cells. Before culturing according to the methods of theinvention the cells are typically undifferentiated cell such as stemcells or pluripotent precursor cells. The cells are typicallynon-specialized cells, or cells that are not mature, or cells that canundergo further stages in development. The shape of the cells beforeculturing is typically amorphous or spherical. The arrangement of thecells before culturing is typically incoherent, or without awell-defined order. The cells which are cultured may be continuous celllines (e.g. with an immortal phenotype), primary cell cultures,transformed cell lines, finite cell lines (e.g. non-transformed cells),or any other cell population maintained in vitro. The cells may beisolated, purified or partially purified cells.

Neurons are preferred cells, and the method of the invention may resultin formation of sympathetic neurons.

The cells are typically responsive to the growth factor. They may be ofthe same species as the growth factor. They may be cells associated withany condition mentioned herein and/or they may be cells which can beused to treat any condition mentioned herein. Whenever ‘treatment’ or‘treating’ is mentioned it includes ‘preventative’ or ‘prophylactic’uses. The cells may be associated with or responsible for any of theactivities mentioned in herein or in Table 1.

Growth Factor

As used herein a growth factor is typically an extracellular proteincapable of stimulating or inhibiting cellular growth, proliferation andcellular differentiation. Thus the growth factor may be a hormone orcytokine. The growth factor may be a natural or artificial one, and mayfor example be a homologue and/or fragment of a natural growth factorwhich retains growth factor activity. It may be a homologue and/orfragment of any specific growth factor mentioned herein, for example inTable 1. It may be a eukaryotic, preferably human or animal, such asmammalian or avian, growth factor.

The growth factor can be a neurotrophin. The expression ‘Neurotrophin’is used interchangeably herein with the expression ‘Neurotrophic growthfactor’. Neurotrophins are the family of biomolecules that support thegrowth, survival and differentiation of both developing and matureneurons. The growth factor can be one or more members of one of thethree main families, such as neurotrophin family (for example, nervegrowth factor NGF, neurotrophin-3 NT-3, brain-derived neurotrophicfactor BDNF, neurotrophin 4 NT-4 (also known as NT-5), ciliaryneurotrophic factor (CNTF) family (CNTF, Leukemia inhibitory factor LIF,Interleukin-6), and GDNF (Glial cell-line neurotrophic factor) family(for example GDNF, CDNF, Artemin, Neuturin, Persephin). The growthfactor may be a semaphorin or slit molecule.

Neurons can be cultured using gradients of neurotrophic growth factors.Nerve growth factor (NGF) can promote myelination and/or differentiationof neurons and/or axon growth and/or dendrite formation and/orelongation of the neuron perpendicular to the direction of the gradientand/or intermediate filament growth and/or tau protein expression and/ormicrotubule growth.

The growth factor can be a bone growth factor. A bone growth factor is agrowth factor that stimulates the growth of bone tissue. Bone growthfactors include bone morphogenetic proteins (BMPs), insulin-like growthfactor (IGF-1), insulin-like growth factor-2 (IGF-2), transforminggrowth factor beta (TGF-b), fibroblast growth factors (FGFs),platelet-derived growth factor (PDGF), parathyroid hormone-relatedpeptide (PTHrP), bone morphogenetic proteins (BMPs), and certain membersof the growth differentiation factor (GDF) group of proteins. PreferredGDF proteins include:

GDF1—Studies in rodents suggest that this protein is involved in theestablishment of left-right asymmetry in early embryogenesis and inneural development in later embryogenesis.

GDF2—This protein regulates cartilage and bone development, angiogenesisand differentiation of cholinergic central nervous system neurons.

GDF5—This protein regulates the development of numerous tissue and celltypes, including cartilage, joints, brown fat, teeth, and the growth ofneuronal axons and dendrites

GDF7—This protein may play a role in the differentiation of tendon cellsand spinal cord interneurons.

GDF10—This promotes neural repair after stroke.

GDF11—This protein plays a role in the development of the nervous andother organ systems, and may regulate aging.

The growth factor is preferably one which exerts an effect viamicrotubules, and or intermediate filaments/and or microfilaments, suchas neuronal growth factors, or neurotrophins, for example NGF.

Table 1 describes preferred growth factors and exemplified constructsfor expressing them, one or more of which may be used in the methods ofthe invention.

The growth factor in the gradient will be in purified or substantiallypurified form.

TABLE 1 GF PODS construct Tag Growth factor Roles include: 1 ActivinA-H1 H1 Activin Cell proliferation, differentiation, apoptosis, H29S ΔCCmetabolism, homeostasis, immune response, wound repair and endocrinefunction 2 BDNF-H1 H1 Brain-derived Growth and differentiation of newneurons H29S ΔCC neurotrophic and synapses factor 3 BMP2-Full-H1 H1 BoneStimulates production of bone H29S ΔCC morphogenetic protein 2 4BMP-4-Full-H1 H1 Bone Differentiation in embryo, bone formation H29S ΔCCmorphogenetic protein 4 5 EGF-H1 H1 Epidermal growth Cellularproliferation, differentiation, and survival H29S ΔCC factor 6Endostatin H1 Endostatin Blocks the proliferation and organization ofB2-H1 endothelial cells into new blood vessels. Inhibits H29S ΔCCangiogenesis and growth of both primary tumours and secondarymetastasis. 7 FGF2-H1 H1 Basic fibroblast FGF family members bindheparin and possess H29S ΔCC growth factor broad mitogenic andangiogenic activities. FGF-2 causes limb and nervous system development,wound healing, and tumour growth. 8 FGF7-H1 H1 Keratinocyte A potentepithelial cell-specific growth factor, H29S ΔCC growth factor whosemitogenic activity is predominantly exhibited in keratinocytes but notin fibroblasts and endothelial cells. Role in morphogenesis ofepithelium, reepithelialization of wounds, hair development and earlylung organogenesis. 9 GDNF-H1 H1 Glial Cell Derived Promotes thesurvival and differentiation of H29S ΔCC Neurotrophic dopaminergicneurons in culture, and prevents Factor apoptosis of motor neuronsinduced by axotomy. 10 IGF-1-H1 H1 Insulin-like Primarily made by theliver as an endocrine H29S ΔCC growth factor 1 hormone as well as intarget tissues in a paracrine/autocrine fashion; produced throughoutlife. The highest rates of IGF-1 production occur during the pubertalgrowth spurt. The lowest levels occur in infancy and old age. 11 IL10-H1H1 Interleukin 10 This cytokine has pleiotropic effects in H29S ΔCCimmunoregulation and inflammation. It down-regulates the expression ofTh1 cytokines, MHC class II Ags, and costimulatory molecules onmacrophages. It also enhances B cell survival, proliferation, andantibody production. 12 IL6-H1 H1 Interleukin 6 Functions ininflammation and the maturation of H29S ΔCC B cells 13 LIF-H1 H1Leukaemia Involved in the induction of hematopoietic H29S ΔCC InhibitoryFactor differentiation in normal and myeloid leukemia cells, inductionof neuronal cell differentiation, regulator of mesenchymal to epithelialconversion during kidney development 14 mEphrin H1 Ephrin B2 Implicatedin mediating developmental events, B2-H1 (species: mouse) especially inthe nervous system and in H29S ΔCC erythropoiesis. 15 NGF-Full-H1 H1Nerve Growth NGF is a neurotrophic factor and neuropeptide H29S Factorprimarily involved in the regulation of growth, maintenance,proliferation, and survival of certain target neurons. In fact, NGF iscritical for the survival and maintenance of sympathetic and sensoryneurons, as they undergo apoptosis in its absence. 16 NGF-Mature-H1 H1H29S ΔCC 17 PDGF-B-H1 H1 Platelet Derived PDGF subunit B, which canhomodimerize, bind H29S ΔCC Growth Factor and activate PDGF receptortyrosine kinases, which play a role in a wide range of developmentalprocesses. 18 Rank-L-H1 H1 Receptor RANK Ligand plays a critical role inbone H29S ΔCC activator of metabolism, particularly osteoclast nuclearfactor differentiation. In addition, RANK Ligand is kappa-B ligandexpressed by some T cells and promotes dendritic cell maturation.Recombinant soluble human RANK Ligand is a non-glycosylated protein. 19Rank-L-VP3 VP3 H29S ΔCC 20 SCF-H1 H1 Stem Cell Factor; Cytokine thatbinds to the c-KIT receptor. SCF can H29S ΔCC KIT Ligand exist both as atransmembrane protein and a soluble protein. This cytokine plays animportant role in hematopoiesis (formation of blood cells),spermatogenesis, and melanogenesis. 21 SHH-H1 H1 Sonic Hedge Hog Memberof a small group of secreted proteins H29S ΔCC that are essential fordevelopment in both vertebrates and invertebrates. 22 TGFb1-H1 H1Transforming The protein regulates cell proliferation, H29S ΔCC GrowthFactor differentiation and growth, and can modulate Beta 1 expressionand activation of other growth factors including interferon gamma andtumor necrosis factor alpha. This gene is frequently upregulated intumor cells. 23 TGFb3-H1 H1 Transforming The protein is involved inembryogenesis and cell H29S ΔCC Growth Factor differentiation, and playsa role in wound healing. Beta 3 24 mVEGF-164-H1 H1 Vascular The proteininduces proliferation and migration H29S ΔCC Endothelial of vascularendothelial cells, and is essential for Growth Factor both physiologicaland pathological angiogenesis. (e.g. in the construct 164 amino acidvariant, species mouse) 25 Wnt3a-H1 H1 Wingless-related A role inoncogenesis and in several H29S ΔCC integration site developmentalprocesses, including regulation of family, member3a cell fate andpatterning during embryogenesis.

Differentiation and/or Proliferation

Differentiation is typically the process whereby cells becomespecialized in order to perform a specific function. Through culturingaccording to the method of the invention cells can alter by acquiringspecialized structural and/or functional features.

Proliferation is typically a process that results in an increase in thenumber of cells. Proliferation can be regulated by the slope (change inconcentration per unit distance) of a gradient and/or by the directionof gradient.

Changes that Typically Occur in the Method of the Invention

The cells may alter in one or more structural features including:

Change of type: Cells can change to a different type in response to agradient of growth factor. Cells can become a first type at one part ofthe gradient. Cells can become a second type at a second part of thegradient. For example for Wnt in mammals at the highest concentrationestablishes the posterior region whilst in areas of lowest concentrationestablishes the anterior region.

Change in arrangement or long range order: The cells can become more orless prevalent at one part of the gradient in comparison to unculturedcells or cultured cells grown in an equivalent uniform concentration ofgrowth factor. The cells can become more or less prevalent at one partof the gradient in comparison to cells grown at a second part of thegradient. In one embodiment the cells are completely absent from certainpoints of the gradient. The cells or prominent cellular features, forexample projections, such as neurites, optionally in axons or dendrites,can align in a direction which is well defined with respect to thedirection of the gradient, for example along the gradient, orperpendicular to the gradient.

In one embodiment the cells align and/or attach to other (for example asdescribed in the Examples). Such cells may align at a distance of 50 to150 microns from the PODS, such as at a distance of 80 to 120 microns.In one embodiment there may be only a single set of aligned and/orattached cells (for example forming a circle), or there may be noaligned and/or attached cells outside the above distance ranges. Thealignment is preferably autonomous, in that the gradient is the onlycause of the alignment.

Change of shape: Cells can change shape optionally becoming elongated inshape, or can alter so that there is an increase in the cell surface tovolume ratio. In an elongated cell the ratio of longest to shortestdimension is typically at least 2 to 1, at least 5 to 1, at least 10 to1, at least 20 to 1, at least 100 to 1, at least 500 to 1, andoptionally less than 100,000 to 1 or less than 10,000 to 1.

Change in expression profile: Cells can upregulate expression ofproteins not expressed or minimally expressed compared to unculturedcells or cultured cells grown in equivalent uniform concentration ofgrowth factor. Cells can downregulate expression of proteins notexpressed or minimally expressed compared to uncultured cells orcultured cells grown in equivalent uniform concentration of growthfactor. Cells can upregulate expression of one or more proteins anddownregulate expression of one or more different proteins not expressedor minimally expressed compared to uncultured cells or cultured cellsgrown in equivalent uniform concentration of growth factor. Expressionof proteins relevant to the structure of the cells may be upregulated,optionally proteins in filaments such as microfilaments, neurofilaments,microtubules, associated with microtubules such as tau. Expression ofreceptors can be upregulated. In one embodiment specific markers areexpressed when the cells are altered.

Change in physical features: A change in the microtubules and/ormicrotubule associated proteins and/or intermediate filaments, forexample tau and neurofilaments can occur, such as alignment of themicrotubules or intermediate filaments, optionally neurofilaments, withrespect to the gradient of growth factor, for example alignment of thelongest dimension of the cell along the gradient of the growth factor oralignment of the shortest dimension of the cell along the gradient ofthe growth factor.

Cells cultured according to the methods of the invention may form lines(e.g. straight, curved or circular lines) of connecting cells,optionally where the line of cells is orientated perpendicular to thedirection of the growth factor gradient.

Nerve cells, or neurons, cultured according to the method of theinvention are preferably elongated in shape, and/or unbranched and/orhave neurite projections at polar positions. Nerve cells may have axonsand/or a growth cone/and or dendrites.

The Gradient

In the method of the invention the growth factor is not present at ahomogenous concentration. Instead it is in the form of a gradient. Asused herein, the gradient is typically the change in the value of growthfactor concentration per unit distance in a particular direction,normally determined by sustained release by the PODS and diffusionthrough the medium. The concentration is preferably highest at the PODSand decreases with distance away from the PODS. The direction of thegradient is from the/a region containing PODS towards the/a regioncontaining no PODS. For a circular arrangement of PODS in 2D or 3D thedirection of the gradient is radially outwards from the PODS. For alinear arrangement of PODS the direction of the gradient isperpendicular to the axis along which the PODS are arranged. Thegradient is provided by the PODS which, for example, break down andrelease growth factor in situ.

Growth factor is typically slowly released from the microcrystals field(which is the location where the PODS are), preferably resulting in asteady physiologically relevant gradient in growth factor at theperiphery of the field. This microenvironment can result in thealteration of the cells in one or more ways as described in theparagraphs above. Typically for neurons, culturing in a growth factorgradient results in alteration of the cells in all of the ways describedin the paragraphs above. The altering for neurons cultured in a growthfactor gradient includes induction of differentiation and can inducealignment of cells, for example nerve cells, preferably PC12 cells.

Gradients will normally occur when the PODS is confined to a small partof the culture space. An important property of a PODS is that ittypically generates short gradients. Short gradients are important forreducing off-target effects caused by high concentrations of growthfactor diffusing to the surrounding area, for example to neighbouringtissues in therapeutic use. The concentration of growth factor in thegradients described herein is typically at a maximum in themicrocrystals field. The concentration reduces as the distance from themicrocrystals field increases, typically dropping to half of the maximumvalue at up to 5 microns, up to 10 microns, or up to 30 microns, or upto 60 microns, or up to 100 microns, or up to 150 microns from themicrocrystals field. The PODS usually provide a ‘short range’ gradient,and in certain situations problems can arise with long range gradients.In one embodiment the gradient has a maximum size of up to 300 microns,such as up to 200 microns.

The gradients of the invention will be established in up to a day. Thegradient may persist/last for at least 12 hours, 1 day, 5 days, 14 days,30 days or 90 days. In a preferred embodiment the gradient isunchanging, or substantially unchanging. For example the concentrationof the growth factor does not decrease by more than 10% over 12 hours,over 1 day or over 2 days at 10 microns from the PODS.

Microcrystals

Viruses such as cypoviruses and baculoviruses produce microcrystalswhich are crystals of the protein polyhedrin, also known as polyhedra.The microcrystals of the invention, or polyhedra, are typically regulararrays of the polyhedrin protein assembled in a cubic crystal lattice.The microcrystals are typically cubic in shape, or optionally form asirregular crystals. The microcrystals of the invention are typicallyisolated from cells, such as insect cells, which have been infected withvirus, preferably baculovirus. The microcrystals typically range in sizefrom 0.1 to 10 micron, for example 2 to 5 micron (measured as themaximum distance inside the microcrystal between different surfaces orthe maximum length of an edge). The microcrystals typically comprise,for example encapsulate, one or more types of growth factor.

PODS and Polyhedra

The PODS used in the invention is typically in the form of, orcomprises, microcrystals which comprise a structural protein and agrowth factor. Each microcrystal (PODS) comprises more than one copy ofthe structural protein and growth factor, and typically comprises 10⁷ to10⁹, for example 10⁸ polyhedron proteins and/or 10⁷ to 10⁹, for example10⁸ growth factor molecules. Typically each PODS comprises 5×10⁷ to5×10⁸ polyhedron proteins and/or 5×10⁷ to 5×10⁸ growth factor molecules.

The structural protein is typically capable of forming polyhedra, and ispreferably a natural or artificial polyhedrin protein. The polyhedrinprotein is typically of viral origin, for example a virus of the genusCypovirus (CPV) or Baculovirus, such as a Bombyx mori cypoviruspolyhedrin. Polyhedra are typically in the form of microcrystals whichare preferably cubic crystals. Optionally PODS used in the methods ofthe invention have a maximum dimension (defined by distance betweensurfaces or length of an edge) of 1 micron, 2 micron, 1-4 micron, up to10 micron, up to 15 micron or up to 20 micron. The polyhedrin proteintypically has homology to all or a part of any such protein describedherein.

Characteristics of the Crystal

Typically a crystal unit cell has a dimension of 100 angstroms, i.e.1×10⁻⁸ m. In a 1 micron crystal, a cube with each edge 10⁻⁶ m long,there are 100 unit cells along each edge, and so a 1 micron crystal has100×100×100 unit cells, i.e. 10⁶ unit cells. Each unit cell typicallycontains 24 copies of polyhedrin, so a 1 μm crystal has 24×10⁶, or2.4×10⁷ copies of polyhedron, a density of 2.4×10⁷ per μm³. That means a2 μm crystal has 2×2×2×2.4×10⁷=19.2×10⁷=approx. 2×10⁸ copies ofpolyhedrin.

H1 tag: this replaces H1 in the polyhedrin. Assuming a 1 in 4incorporation to minimise disrupting the crystal (H1 forms bunches offour), this would give 6 copies of H1-Growth factor per micron crystal,6×10⁶ per μm³, and so typically so a 1 μm³ sized PODS contains 6×10⁶ perμm³ copies of growth factor.

VP3 tag: potentially 1 VP3 binding site per polyhedrin molecule, so upto 2.4×10⁷ per μm³, and 2.4×10⁷ copies in a 1 μm crystal.

Typically a crystal comprises 10⁷ to 10⁹ molecules of growth factor,such as about 10⁸ molecules.

Attaching, Encapsulating or Packaging Growth Factor

The PODS of the invention comprises growth factor. This is typicallyimmobilised and/or attached to the PODS in manner that allows release toprovide the gradient. In a preferred embodiment the growth factorcomprises a tag that is used to attach it to the PODS.

The growth factor can be targeted for packaging in a polyhedra byattachment of the tag to the growth factor. The growth factorpolypeptide can typically comprise the growth factor amino acid sequenceand the amino acid sequence of part of the polyhedrin protein,preferably helix 1 (H1), even more preferably a sequence with at least80% homology to an H1 sequence mentioned herein. The growth factorpolypeptide can comprise the growth factor amino acid sequence and theamino acid sequence of part of the baculovirus coat protein, preferablyVP3, even more preferably a sequence with at least 80% homology to a VP3sequence disclosed herein. A short VP3 sequence is preferred to minimiseany disruptive effect on the PODS.

PODS incorporating growth factor can be prepared by coexpressingpolyhedrin protein and polypeptide comprising a targeting portion and agrowth factor portion. Cells can be inoculated with recombinantbaculovirus expressing polyhedrin to generate empty polyhedra. Cells canbe coinfected with recombinant baculovirus expressing polyhedrin andrecombinant baculovirus expressing a polypeptide comprising the growthfactor and a tag which targets the growth factor for packaging, forexample VP3 or H1. PODS comprising polyhedrin and the polypeptide can beprepared. Cells can be coinfected with recombinant baculovirusexpressing polyhedrin and one or more different recombinantbaculoviruses each expressing a different polypeptide comprising thegrowth factor and a tag which targets the growth factor for packaging.

PODS comprising polyhedrin and one or more different polypeptides,encoding one or more different growth factors, can be prepared. Thedifferent polypeptides can have the same tag or different tags.

The polyhedrin protein can be Bombyx mori cypovirus polyhedrin protein.The recombinant baculovirus can be AcCP-H29 expressing Bombyx moricypovirus polyhedrin. The cells used to produce the PODS, or growthfactor-encapsulated polyhedral, can be Spodoptera frugiperdaIPLB-SF21-AE cells (Sf cells). The PODS can be isolated from the cells,optionally insect cells, by centrifugation.

The PODS can be prepared in an aqueous suspension. The concentration ofthe PODS suspension can be more than 10⁴ PODS per microliter volume,typically from 10⁴ to 10⁷ PODS per microliter volume, preferably from5×10⁴ to 5×10⁶ or 10⁵ to 10⁶ PODS per microliter volume. PODS can beapplied to (contacted with) the culture system/medium at theseconcentrations. These can be used in therapy at concentrations of 10⁵ to10⁷ crystals per microliter, for example at 3×10⁶ crystals permicroliter.

The Microcrystals (PODS) Field

The microcrystals field is the location of the microcrystals (that setup the gradient). The term ‘microcrystals field’ includes the feature ofa ‘PODS field’ where the PODS are not in crystalline form.

The geometry and size of the microcrystals field may depend on thegrowth factors. The microcrystals field can be a circular, linear orrectangular field. The shape of the field can be customized for aparticular use, for example it can be a curved, including S-shaped, orit can be a shape consisting of straight lines, such as a square,hexagon or an octagon. The microcrystals field can comprise 10⁴-10⁸,preferably 10⁵-10⁷ or 5×10⁵-10⁶ microcrystals in total, or this range ofmicrocrystals in 20 mm². The field can comprise up to 10⁵ microcrystals,or up to 10⁶ microcrystals, for example in 20 mm². The microcrystalsfield can comprise one or more shape elements, for example one or morelines. Each shape element can comprise these stated ranges ofmicrocrystals, for example 10⁴-10⁵ microcrystals, or up to 10⁵microcrystals, or up to 10⁶ microcrystals.

A microcrystals field can comprise one or more lines or shapes. Thelines can be straight lines. The lines and/or shapes may be spaced sothat the concentration of growth factor reaches substantially zerobetween the lines and/or shapes. Adjacent lines or shapes may eachcomprise the same growth factor. Adjacent lines or shapes may eachcomprise a different growth factor. The lines and/or shapes mayintersect. For example, the lines may form a regular grid pattern. Oneor more lines or shapes may comprise one or more different growthfactors. In a preferred embodiment the field is a 0.5 to 10 mm diameter,for example 1 to 8 or 2 to 4 mm diameter circular field.

A grid structure is envisaged in which individual lines of the grid cancontain different growth factor combinations. Adjacent and intersectinggrid lines can provide areas where complex gradients will develop,allowing the creation of a wide range of defined and addressable cultureconditions in a small area. The lines of the grid can be placed up to0.1 mm, up to 1 mm, up to 5 mm, from 0.1 mm to 1 mm, from 1 mm to 5 mm,apart.

In the methods herein, the microcrystals field may be applied to asurface, for example the surface of a coverslip, the surface of a cellculture vessel, or the surface of a delivery vehicle such as hydrogel.The microcrystals field may be applied by hand, for example using amicropipette, or may be applied using a robot or other methods ofsurface deposition including lithography. The microcrystals field can bedried after application to a surface, for example for more than 3 hours.The microcrystals field may be extruded into hydrogel for example usinga syringe. The use of a hydrogel or other carrier can increase theduration of the gradient. Synthetic hydrogels or natural materials,preferably collagen, can be used as a delivery vehicle. A field of PODSas described herein can be provided in a delivery vehicle such as alayer of hydrogel which can be manipulated, for example to provide agradient of growth factor in a defined direction along a pre-determinedpath. For example, in one embodiment the methods provide an insulatedline of PODS spanning an injury to encourage axons to navigate acrossthe injury.

For neuronal growth factors a linear pattern is envisaged. There are anynumber of different geometries that may be effective. One possibility iscreating 3D sandwiches of cells and PODS. A microcrystal fieldcomprising parallel lines of PODS (0.1 to 1 mm apart) can be created. Alayer of soft hydrogel or other delivery system vehicle could be layeredon this and optionally seeded with neuronal cells or neuronal precursorcells. A stiffer layer of hydrogel can be layered above. After the gelhas set, the gel can be manipulated to change the 3D disposition of thelines of PODS. For example, the structure could be rolled to create a“Swiss roll” of cells and PODS. The nerve cells would form linear,unbranched connections perpendicular to the gradient, and thereforealong the lines of PODS. This entire structure comprising PODS and cellscould be used as an implant, for example to restore nerves that had beenlost due to neurodegeneration or injury.

PODS are typically denser than water, and can sink to the bottom ofaqueous cell culture medium in a few seconds to within 10 minutes. PODSmay be held in place in a culture system by gravity. The microcrystalsmay be added to a drop of cell culture medium suspended from a surface,optionally example a plastic surface, for example a coverslip. Thiswould establish a gradient across the drop with cells at the bottomexperiencing the highest concentrations.

Any suitable may be used to place or attach the PODS. In one embodimentthe PODS is attached via a micropatterning system, for example using aphotoactivatable reagent and a UV illumination system.

Embodiments

In one embodiment the method of the invention does not comprise acomponent that mimics an extracellular matrix, for example it might notcomprise collagen gel compaction, an electromagnetic field,electrospinning of cells, mechanical stimulation and microstructuredculture plates.

For the avoidance of doubt any feature described herein, such asspecific growth factors or specific properties of cells may be excludedin certain embodiments of the invention.

Medical Use

The invention includes setting up a gradient for use in therapy.Therefore the invention includes administering the PODS for preventingor treating a condition, such as any condition mentioned herein.

Preferred conditions include growth factor deficiency, degeneration,injury or nerve damage. The PODS may be used in the manufacture of amedicament for preventing or treating a condition. Thus the PODS may beused in therapy, optionally together with a delivery vehicle, forexample collagen or a gel (such as a hydrogel). The PODS may bedelivered using a scaffold. Examples of diseases that can be treated bythe PODS are mentioned herein, including in Table 1.

In preferred embodiments:

-   -   PODS for therapy of ALS can comprise IGF-1.    -   PODS for therapy of Parkinson's can comprise GDNF, GDF-5, and/or        CDNF.    -   PODS for cartilage repair can comprise the TGFb family.    -   PODS for therapy of multiple sclerosis can comprise LIF.    -   PODS for therapy of bone fracture can comprise bone        morphogenetic protein 2 or bone morphogenetic protein 4    -   PODS for therapy of ALS or cardiac conditions can comprise VEGF.

Cells prepared using the method of the invention may be used in therapy,for example to treat any condition mentioned herein, such asdegeneration, injury or nerve damage. In a preferred embodiment nervecells prepared in a gradient of neurotrophic growth factors, can be usedin a method of treatment for nerve injury or for neurodegeneration.

Homologues and Fragments

Homologues of polypeptide sequences are referred to herein (for examplegrowth factors and tags). Such homologues typically have at least 70%homology, preferably at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98% or at least 99% homology, for exampleover a region of at least 10, 15, 20, 30, 100 or more contiguous aminoacids. The homology may be calculated on the basis of amino acididentity (sometimes referred to as “hard homology”). Preferredhomologues retain activity, for example growth factor or tag activity.

The UWGCG Package provides the BESTFIT program which can be used tocalculate homology and/or % sequence identity (for example used on itsdefault settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculatehomology and/or % sequence identity and/or line up sequences, such asidentifying equivalent or corresponding sequences (typically on theirdefault settings), for example as described in Altschul S. F. (1993) JMol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pair (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as theneighbourhood word score threshold (Altschul et al, supra). Theseinitial neighbourhood word hits act as seeds for initiating searches tofind HSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W5 T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both sequences.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between twopolynucleotide sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to the second sequenceis less than about 1, preferably less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

The homologous sequence typically differs by 1, 2, 3, 4 or more aminoacids, such as less than 10, 15 or 20 amino acids (which may besubstitutions, deletions or insertions of amino acids). These changesmay be measured across any of the regions mentioned above in relation tocalculating homology and/or % sequence identity.

Fragments of any of the polypeptides mentioned herein can be used.Typically such fragments retain the original activity, for examplegrowth factor or tag activity. The fragments typically comprise at least60%, for example at least 70%, 80%, 90% or 95% of the original sequence.

Therapeutic Agents

Therapeutic agents (PODS and cells) and uses are mentioned herein. Theinvention provides such agents for use in preventing or treating therelevant condition. This may comprise administering to an individual inneed a therapeutically effective amount of the agent. The inventionprovides use of the agent in the manufacture of a medicament to preventor treat the disease.

The formulation of the agent will depend upon the nature of the agent.The agent will be provided in the form of a pharmaceutical compositioncontaining the agent and a pharmaceutically acceptable carrier ordiluent. Suitable carriers and diluents include isotonic salinesolutions, for example phosphate-buffered saline. The agent may beformulated for parenteral, intravenous, intramuscular, subcutaneous,transdermal or oral administration. Preferably administration can be byinjection, nasal (a spray or dry powder), by inhaler or as eye drops.

The dose of an agent may be determined according to various parameters,especially according to the substance used; the age, weight andcondition of the individual to be treated; the route of administration;and the required regimen. A physician will be able to determine therequired route of administration and dosage for any particular agent. Asuitable dose may however be from 0.1 to 100 mg/kg body weight such as 1to 40 mg/kg body weight, for example, to be taken from 1 to 3 timesdaily.

EXAMPLES Example 1. Generic Method for Construction, Expression andPurification of Growth Factor Polyhedra of Table 1

The requisite cDNA for selected growth factors (see Table 1) waspurchased from commercial suppliers. Using standard cloning techniques,growth factors sequences were cloned and subsequently subcloned intotransfection the plasmids pDEST/VP3 and/or pDEST/H1), resulting in theproduction of transfer vectors encoding a growth factor sequence fusedto VP3 or H1 tags, C-terminally or N-terminally, respectively. Then,Spodoptera frugiperda cells were co-transfected with growth factortransfection vectors and a linear version of non-recombinant parentbaculovirus DNA. The successful homologues recombination within insectcells inserted growth factor sequences into the baculovirus genome andso created a recombinant baculovirus variant that allowed theco-expression of a tagged growth factor protein and the polyhedrinprotein, both under the control of the polyhedrin promoter for maximumefficiency.

Recombinant baculovirus was amplified as required and growth factorpolyhedra were subsequently expressed in suspension insect cell culturesusing a shaking incubator for 7-10 days at 27° C. The rapid progress ofthe cubic growth factor polyhedra expression was monitored bymicroscope. After culturing for 7-10 days, polyhedra containing cellswere harvested by centrifugation and cell pellets stored at −20° C.

Growth factor polyhedra were isolated from insect cells by cell lysisand next purified by at least 3 rounds of PBS washes (from commercialsuppliers, pH 7.6-7.9), followed by centrifugation at 3000×g, 4° C., oruntil a pure product has been achieved, where pure polyhedrin ischaracterised by a milky-white appearance in aqueous liquid, dependingon the concentration of polyhedra, and was readily assessed under astandard bright field microscope with a 20× and 40× magnification.Lastly, growth factor polyhedra were counted and stored at 4° C. in PBS(pH 7.6-7-9).

Example 2. Construction of NGF Polyhedra

The cDNA encoding the NGF ORF was purchased from Toyobo in an entryvector. The full-length (241 amino acids) and mature (120 amino acids)form NGF were cloned and subcloned into each destination vector(pDEST/VP3, and pDEST/H1), resulting in production of the transfervectors encoding the full-length or mature form of NGF fused with VP3 orH1 tags (pTransH1/full NGF, pTransH1/mature NGF, pTransVP3/full NGF, andpTransVP3/mature NGF). These transfer vectors were co-transfected intoSf21 and/or Sf9 insect cells with BaculoGold™ baculovirus linearizedDNA. After incubation for 5 days at 27° C., recombinant baculovirusesexpressing the full-length and mature forms of NGF with VP3 or H1 tagswere harvested and stored at 4° C.

Full-length or mature NGF was fused with polyhedron-targeting tags (H1or VP3) to obtain NGF-encapsulated polyhedra (PODS NGF) (FIG. 1).

Example 3. Purification of NGF-Encapsulated Polyhedra

To generate empty polyhedra, Spodoptera frugiperda IPLB-5F21-AE cells(Sf cells) were inoculated with recombinant baculovirus AcCP-H29Sexpressing Bombyx mori cypovirus polyhedrin under the control of thebaculovirus polyhedrin promoter. For production of NGF polyhedra (PODSNGF), Sf cells were co-infected with AcCP-H29S and another recombinantbaculovirus expressing recombinant NGF fused with the VP3 or H1 tags.The infected cells were cultured for 10 days at 27° C. and then thecells were harvested in a conical tube by centrifugation. The cellpellet was resuspended in phosphate-buffered saline (PBS; pH 7.2) andtreated with an ultrasonic homogenizer at 6% power for 30 sec. The cellhomogenate was centrifuged at 1500×g at 4° C. and the supernatant wasremoved. These treatments were repeated and the purification wascomplete. The polyhedron suspension was adjusted to 5×10⁴ or 1×10⁵numbers per microliter volume and stored at 4° C. in distilled watercontaining 100 units/ml penicillin and 100 μg/ml streptomycin.

Example 4. Methods for Preparing a Gradient

-   -   a. Circular patch: Cover slip coating. Cover slips were        sterilized with 70% ethanol and subsequently washed with        sterilized water. Cellmatrix Type IV was dissolved in 0.15 M        acetic acid (30 μg/mL). Sterilized cover slips were put and 0.5        mL of Cellmatrix Type IV solution was then added to culture        wells. Following overnight incubation, the Cellmatrix Type IV        solution was discarded and the cover slips were allowed to dry        for 1 h. Collagen-coated cover slips were washed twice with        serum-free DMEM. One microliter of polyhedron suspension (5×10⁴        or 1×10⁵ numbers/μl) was spotted on the cover slips and allowed        to dry for more than 3 h.

Example 5. Culturing NGF

One microliter of PODS NGF suspension (5×10⁴ or 1×10⁵ crystals/μl) wasmanually spotted onto a gelatin-coated cover slip using a micropipetteto create a circular field of approximately 2-4 mm in diameter of eitherPODS full-length NGF or PODS mature NGF (FIG. 1). PC12 cells were seededand cultured with serum-free medium under static conditions for 96 hourswithout media change. At the end of this period, alignment of PC12 cellsalong the periphery of the PODS fields was observed. PODS full-lengthNGF prominently induced axon extension from each cell. This axonextension was parallel with the edge of the PODS field resulting in theformation of a connected chain of migrated cells.

Example 6. Observation of Cells by SEM Imaging

Alignment and axon extension of PC12 cells surrounding PODS NGF fieldwas also observed by scanning electron microscopy (SEM).

PC12 Cell Culture.

The PC12 DMEM cell culture medium was discarded and 0.02% EDTA solutionwas added. After the EDTA solution was discarded, 1 ml of 0.25%trypsin-EDTA was added and incubated for 1 min. One millilitre of DMEMculture medium was added and the cell suspension was centrifuged at1,200 rpm for 2 min. After the supernatant was discarded, cells weresuspended in 1 ml of serum-free DMEM and the number of cells wascounted. Cells (7×10⁴ cells/well) were then seeded into a well. Half ofthe medium (serum-free DMEM) was gently exchanged after 3 days. Imagesof cell alignment were obtained via scanning electron microscopy (SEM)on the 5th day following cell seeding.

Preparation of Cells for SEM Imaging.

After the cell medium was gently discarded, cells were fixed by 4%paraformaldehyde phosphate buffer solution, 1% osmium tetroxide and 1%tannic. Dehydration was carried out by immersing the cover slips in aseries of ethanol solutions of increasing concentrations until 100%dehydration was achieved. Cover slips were covered withhexamethyldisilazane and allowed to dry overnight. Images of cellalignment were obtained via SEM after gold-sputtering (200 Å).

Immunocytochemistry.

For immunofluorescence, cells on the cover slips were fixed for 30 minat room temp with 4% paraformaldehyde. After three washes with PBS for 5min each, the cells were permeabilized with 0.3% Triton X-100 in PBS for15 min at room temperature. After three washes with PBS for 5 min each,the cells were incubated in blocking buffer (3% FBS in PBS) for 1 h atroom temperature and then incubated with primary antibodies (anti-tauantibody (Merck Millipore) or anti-neurofilament heavy polypeptideantibody (SIGMA)) for overnight at 4° C. After three washes with PBS for10 min each, the cells were incubated with FITC-conjugated secondaryantibody (goat anti-mouse IgG antibody (Invitrogen)) for 1 h at roomtemperature. Cover slips were then washed three times with PBS andfinally mounted on microscope slides in mounting medium with propidiumIodide (Invitrogen) for nuclei staining. Stained cells were observedusing an Olympus Fluoview FV1000-IX81 confocal microscope.

These results indicate that PODS NGF field regulates the direction ofalignment and axon extension of PC12 cells. We confirmed thedifferentiation of PC12 cells using tau and neurofilament antibodies.Tau and neurofilament proteins were observed in the aligned PC12 cells(FIG. 2a ). Neurofilament was notably detected extending from theextended axon, indicating that the aligned cells are differentiated tonerve cells. A helical structure of neurofilament was also observed inthe neighborhood of the tip of the extended axons. In contrast,expression of tau and neurofilament was not observed in PC12 cellsincubated with empty polyhedra. In some cases the connections of PC12cells were seen to be formed between the extended axon and the growthcone-like structure which were induced by PODS H1/full NGF (a solid boxin FIG. 2b ), but in other cases the connection was not observed (adotted box in FIG. 2b ).

Example 7 Direct Visualisation of a Gradient

To determine the extent of the gradient, PODS NGF were mixed withpolyhedra encapsulating enhanced green fluorescent protein (PODS EGFP),and subsequently spotted on a cover slip. PC12 cells were then seededand incubated with PODS NGF and PODS EGFP, and the fluorescence emissionon the periphery of the aligned PC12 cells was measured (FIG. 3). Agradient of green fluorescence was observed from the polyhedra field.

DNA and Protein Sequences NGF (mature) sequence:TCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGT GAGAAGAGCCTGASSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWR FIRIDTACVCVLSRKAVRRA*H1-NGF fusion protein sequenceatggcagacgtagcaggaacaagtaaccgagactttcgcggacgcgaacaaagactattcaatagcgaacaatacaactataacaAcagcAAGAATTCTAGACCATCAACAAGTTTGTACAAAAAAGCAGGCTCCTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAGAGCCTGAMADVAGTSNRDFRGREQRLFNSEQYNYNNSKNSRPSTSLYKKAGSSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRID TACVCVLSRKAVRRA*NGF-VP3 fusion protein sequenceATGTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAGAGCCATGGGTCGAAAGAACATGTTTCACCATGATGGGTACCTTCTAGCTTTCAACTCACAACGACGATCACACACGTTACGACTACTAGGGCCTTTTCAGTACTTCAACTTCTCCGAGACAGATAGAGGACATCCATTATTTCGCCTACCTCTTAAGTATCCATCAAAAGCAATACCAGCAGATGAGTTAATTGACAATTTACACTAGTAACGGCGGAATAAMSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRAMGRKNMFHHDGYLLAFNSQRRSHTLRLLGPFQYFNFSETDRGHPLFRLPLKYPSKAIPADELIDNLH* NGF (full length) sequence:GAACCACACTCAGAGAGCAATGTCCCTGCAGGACACACCATCCCCCAAGTCCACTGGACTAAACTTCAGCATTCCCTTGACACTGCCCTTCGCAGAGCCCGCAGCGCCCCGGCAGCGGCGATAGCTGCACGCGTGGCGGGGCAGACCCGCAACATTACTGTGGACCCCAGGCTGTTTAAAAAGCGGCGACTCCGTTCACCCCGTGTGCTGTTTAGCACCCAGCCTCCCCGTGAAGCTGCAGACACTCAGGATCTGGACTTCGAGGTCGGTGGTGCTGCCCCCTTCAACAGGACTCACAGGAGCAAGCGGTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAGAGCCTGAEPHSESNVPAGHTIPQVHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA* H1-NGF (full length) fusion protein sequence:atggcagacgtagcaggaacaagtaaccgagactttcgcggacgcgaacaaagactattcaatagcgaacaatacaactataacaAcagcAAGAATTCTAGACCATCAACAAGTTTGTACAAAAAAGCAGGCTCCGAACCACACTCAGAGAGCAATGTCCCTGCAGGACACACCATCCCCCAAGTCCACTGGACTAAACTTCAGCATTCCCTTGACACTGCCCTTCGCAGAGCCCGCAGCGCCCCGGCAGCGGCGATAGCTGCACGCGTGGCGGGGCAGACCCGCAACATTACTGTGGACCCCAGGCTGTTTAAAAAGCGGCGACTCCGTTCACCCCGTGTGCTGTTTAGCACCCAGCCTCCCCGTGAAGCTGCAGACACTCAGGATCTGGACTTCGAGGTCGGTGGTGCTGCCCCCTTCAACAGGACTCACAGGAGCAAGCGGTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAG AGCCTGAMADVAGTSNRDFRGREQRLFNSEQYNYNNSKNSRPSTSLYKKAGSEPHSESNVPAGHTIPQVHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFI RIDTACVCVLSRKAVRRA*NGF-VP3 (full length) fusion protein sequence:ATGGAACCACACTCAGAGAGCAATGTCCCTGCAGGACACACCATCCCCCAAGTCCACTGGACTAAACTTCAGCATTCCCTTGACACTGCCCTTCGCAGAGCCCGCAGCGCCCCGGCAGCGGCGATAGCTGCACGCGTGGCGGGGCAGACCCGCAACATTACTGTGGACCCCAGGCTGTTTAAAAAGCGGCGACTCCGTTCACCCCGTGTGCTGTTTAGCACCCAGCCTCCCCGTGAAGCTGCAGACACTCAGGATCTGGACTTCGAGGTCGGTGGTGCTGCCCCCTTCAACAGGACTCACAGGAGCAAGCGGTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAGAGCCATGGGTCGAAAGAACATGTTTCACCATGATGGGTACCTTCTAGCTTTCAACTCACAACGACGATCACACACGTTACGACTACTAGGGCCTTTTCAGTACTTCAACTTCTCCGAGACAGATAGAGGACATCCATTATTTCGCCTACCTCTTAAGTATCCATCAAAAGCAATACCAGCAGATGAGTTAATTGACAATTTACACTAGTAACGGCGGAATAAMEPHSESNVPAGHTIPQVHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRAMGRKNMFHHDGYLLAFNSQRRSHTLRLLGPFQYFNFSETDRGHPLFRLPLKYPSKAIPADELIDNLH* Bombyx mori polyhedrin sequence (GenBank acc: D37771.1):atggcagacgtagcaggaacaagtaaccgagactttcgcggacgcgaacaaagactattcaatagcgaacaatacaactataacaacagcttgaacggagaagtgagcgtgtgggtatacgcatattactcagacgggtctgtactcgtaatcaacaagaactcgcaatacaaggttggcatttcagagacattcaaggcacttaaggaatatcgcgagggacaacacaacgactcttacgatgagtatgaagtgaatcagagcatctactatcctaacggcggtgacgctcgcaaattccactcaaatgctaaaccacgcgcgatccagatcatcttcagtcctagtgtgaatgtgcgtactatcaagatggccaaaggtaacgcggtatccgtgcccgatgagtacctacagcgatctcacccatgggaagcgaccggaatcaagtaccgcaagattaagagagacggggaaatcgttggttacagccattacttcgaactaccccacgaatacaactccatctccctagcggtaagtggtgtacataagaacccatcatcatacaatgtcggatcagcacataacgtaatggacgtcttccaatcatgcgactcggctctcagattctgcaaccgctactgggccgaactcgaattggtgaaccactacatttcgccgaacgcctacccatacctctatatcaacaatcatagctatggagtagctctgagtaaccgtcagcgattg ctcgtgtaaMADVAGTSNRDFRGREQRLFNSEQYNYNNSLNGEVSVWVYAYYSDGSVLVINKNSQYKVGISETFKALKEYREGQHNDSYDEYEVNQSIYYPNGGDARKFHSNAKPRAIQIIFSPSVNVRTIKMAKGNAVSVPDEYLQRSHPWEATGIKYRKIKRDGEIVGYSHYFELPHEYNSISLAVSGVHKNPSSYNVGSAHNVMDVFQSCDSALRFCNRYWAELELVNHYISPNAYPYLYINNHSYGVALSNRQRL LV* H1-tag sequenceMADVAGTSNRDFRGREQRLFNSEQYNYNNS VP3-tag sequenceAFNSQRRSHTLRLLGPFQYFNFSETDRGHPLFRLPLKYPSKAIPADELID NLH

1. A method of altering cells comprising culturing them in a growthfactor gradient, wherein said gradient is provided by a polyhedradelivery system (PODS) that releases the growth factor to set up thegradient.
 2. The method according to claim 1 wherein said altering is acellular response to the gradient, wherein said cellular responsecomprises: a) growth of cytoskeletal structural components in a defineddirection relative to the direction of the gradient, and/or b)proliferation of cells within a particular concentration range of thegradient, and/or c) different responses in different parts of the cellin response to different concentrations of growth factor cause by thegradient, and/or d) a different branching pattern caused by the presenceof the gradient.
 3. The method according to claim 2 wherein a) saidgrowth is growth of microtubules/and or intermediate filaments and/ormicrofilaments along the direction of the gradient, and/or b) saidgrowth is growth of microtubules/and or intermediate filaments and/ormicrofilaments perpendicular to the direction of the gradient, and/or c)said proliferation of cells is proportional to the concentration ofgrowth factor across the gradient, and/or d) said proliferation isrestricted to a concentration zone in the gradient and which extendsperpendicular to the direction of the gradient, and/or e) saidproliferation is proportional to the slope (change in concentration ofgrowth factor per unit distance) of the gradient.
 4. The methodaccording to claim 1 wherein a) said cells are neuronal cells, and/or b)said PODS releases a growth factor that causes neuron differentiation,and is preferably neurotrophic growth factor.
 5. The method according toclaim 1 wherein said altering is differentiation of the cells and/orwherein said altered comprises a change in: (i) cell type, and/or (ii)cell shape, and/or (iii) protein expression profile.
 6. The methodaccording to claim 1, (i) wherein said altering comprises a change inthe microfilament and/or intermediate filament structure, and/or (ii)said altering causes: a) microfilament and/or intermediate filamentgrowth, preferably tau and/or neurofilament growth, optionally in adirection defined by the gradient; and/or b) neurite projections atpolar positions; and/or c) an unbranched chain connected by axons;and/or (iii) wherein said growth factor is neurotrophic growth factorand said altering comprises neurite outgrowth in a directionperpendicular to the gradient.
 7. The method according to claim 1wherein said PODS comprises a polyhedrin protein and growth factor, andwherein optionally: said growth factor is attached to the polyhedrinprotein, and/or said growth factor comprises a tag, wherein optionallysaid tag targets the growth factor for packaging within polyhedrincrystals in the PODS.
 8. The method according to claim 1 wherein saidPODS is produced by contacting the growth factor with polyhedrin in amethod comprising: contacting microcrystals comprising polyhedrinprotein with tagged growth factor protein, or growing microcrystalscomprising polyhedrin protein in the presence of tagged growth factorprotein.
 9. The method according to claim 1 wherein said polyhedroncomplex is produced in cells co-infected with: a virus expressingpolyhedron, and a virus expressing a growth factor, optionallycomprising a tag, wherein optionally said cells are insect cells, morepreferably SP12 cells, and optionally wherein said polyhedrin is aBaculovirus or Cypovirus polyhedrin, preferably Bombyx mori cypoviruspolyhedrin.
 10. The method according to claim 1 wherein said growthfactor comprises a polypeptide comprising (i) full length NGF accordingto SEQ ID 1 or mature NGF according to SEQ ID 2 or homologues and/orthereof with at least 70% sequence identity to SEQ ID 1 or SEQ ID 2, and(ii) a targeting portion comprising HI (SEQID 3) or VP3 (SEQID 4) orhomologues and/or fragments thereof with at least 70% sequence identityto SEQID 3 or SEQID
 4. 11. A PODS according to claim 1 for use in amethod of treatment of the human or animal body by therapy, wherein saidPODS preferably comprises neuronal growth factor.
 12. The PODS accordingto claim 11 for use in a method of treatment of growth factordeficiency, degeneration, injury or nerve damage.
 13. Cells altered bythe method of claim 1 for use in a method of treatment of the human oranimal body by therapy, preferably for use in treating a growth factordeficiency, degeneration, injury or nerve damage.